Abstract Chronic stress is associated with enhanced visceral pain perception (visceral hyperalgesia) in the human and animal models. An example of chronic stress-associated visceral hyperalgesia in the clinic is Irritable Bowel Syndrome, the most common outpatient diagnosis in Gastroenterology, affecting 10-15% of the general population. The pathways, cellular and molecular mechanisms underlying chronic stress-induced visceral pain are an active area of investigation without a potentially unifying mechanism to explain the pathogenesis, although alterations in the Hypothalamic-Pituitary-Adrenal axis are generally accepted as a contributing factor. Recent studies suggest that chronic stress is associated with impaired intestinal barrier function, increased epithelial paracellular permeability to macromolecules and visceral hyperalgesia. It is unknown whether impaired intestinal barrier function is a prerequisite for the development of chronic stress-induced visceral hyperalgesia. It is also unknown whether chronic stress directly or indirectly alters epithelial tight junction protein expression and, thereby, increases intestinal permeability culminating in activation of nociceptive pathways. This R21 application examine the provocative hypothesis that chronic stress-induced impairment in intestinal epithelial tight junction protein expression and function is a prerequisite for development of visceral hyperalgesia. Down-regulation involving specific intestinal epithelial tight junction proteins is mediated by stress-induced increase in pro-inflammatory cytokine(s) resulting in increased paracellular permeability, and activation of primary afferent nociceptive pathways. Strong preliminary data supports the validity of this hypothesis. Specific Aim 1 will examine a potential causal role between increases in intestinal epithelial paracellular permeability and enhanced visceral pain perception in chronic stress. We hypothesize that chronic stress is associated with impaired intestinal barrier function that precedes visceral hyperalgesia and involves increased permeability to both charged and uncharged macromolecules. Specific Aim 2 will elucidate the molecular pathway that underlies chronic stress-mediated impairment in intestinal barrier function and hyperalgesia. We hypothesize that chronic stress causes elevation in specific pro-inflammatory cytokines, including IL-6, which precedes and mediates down-regulation of specific intestinal epithelial tight junction proteins, increase in macromolecular permeability and consequently visceral hyperalgesia. We propose that both elevation of pro-inflammatory cytokine(s) and increased intestinal (colon) epithelial paracellular permeability to macromolecules are required to produce visceral hyperalgesia. We believe that the data generated with the R21 will form the foundation of a highly competitive R01 application.